Analysis of nucleic acid sequences is being used more and more frequently to determine the xe2x80x9cgenotype,xe2x80x9d or specific type of a particular genetic element, for a nucleic acid sample. Because variant genetic elements exist, techniques have been developed to compare homologous nucleic acids to determine if they are identical, or if they differ by one or more nucleotides. The available genotyping techniques have disadvantages that limit their widespread use and/or efficacy. For example, restriction fragment length polymorphism (RFLP)-based analysis requires large amounts of sample DNA, and has a low density of mapped polymorphisms; furthermore, the number of alleles at each locus is small, leading to a low heterozygosity and therefore an inability to differentiate or distinguish between the homologous nucleic acids a high percentage of the time (Southern, E. M., J Mol. Biol. 98:503-517 (1975); Botstein, D. et al., Am. J Hum. Gen. 32:314-331 (1980)). Short sequence repeat (SSR)-based analysis, which typically utilizes polymerase chain reaction (PCR), reduces the amount of DNA required in comparison with RFLP analysis; furthermore, there are usually many different alleles at each locus leading to high heterozygosity and thus more informative analyses (Miesfeld. R. et al, Nucl. Acids Res. 9:5931-5947 (1987); Weber, J. L., Genomics 7:524-530 (1990)). However, SSR analysis is traditionally a gel-based technique, and is therefore a slow and labor-intensive method: running and reading of gels is expensive, time consuming, error prone and difficult to automate. Because these techniques are being used in an increasing number of fields, such as genetic disease diagnosis, infectious disease diagnosis, forensic identification, paternity determination, and genetic disease or locus mapping, a need exists for simple and efficient methods of genotyping and genetic analysis.
The present invention pertains to assays for analyzing a sample comprising a nucleic acid of interest. In the assays, an array comprising a set of oligonucleotides attached to a solid support is used. Each oligonucleotide in the set comprises a first boundary sequence, a second boundary sequence, a nucleic acid marker, and a label. The first boundary sequence is at the proximal end of the oligonucleotide, relative to the solid support to which the oligonucleotide is attached; the second boundary sequence and the label are at the distal end of the oligonucleotide, relative to the solid support to which the oligonucleotide is attached; and the nucleic acid marker is between the first and the second boundary sequence. The boundary sequences are invariant, and the nucleic acid marker includes the polymorphic region to be assayed.
In one embodiment, the nucleic acid marker is a number N of consecutive repeats of a nucleic acid sequence element. In this embodiment, each nucleic acid marker in the set of oligonucleotides has a different number of consecutive repeats of the nucleic acid sequence element, the number ranging from N to N+A, inclusive. The minimum number N of repeats is generally between 0-40 repeats; the range number (A) is generally between 3 and 20. In another embodiment, the nucleic acid marker contains a single nucleotide polymorphism; each nucleic acid marker in the set of oligonucleotides has a nucleic acid marker that contains a different nucleotide at a polymorphic nucleotide site. In a third embodiment, the nucleic acid marker contains allele-specific polymorphisms; each nucleic acid marker in the set of oligonucleotides contains a different historically observed allele-specific polymorphism. In a fourth embodiment, the nucleic acid marker contains a fragment of the nucleic acid of interest; each nucleic acid marker in the set of oligonucleotides has a different fragment of the nucleic acid of interest, such that the entire nucleic acid of interest is represented by the nucleic acid markers in the set of oligonucleotides.
In the assays, the oligonucleotides are attached to a solid support at identifiable locations, forming the array. The set of oligonucleotides is attached such that the composition and position of each oligonucleotide on the array is identifiable. The array is contacted with a test sample that contains the nucleic acid of interest, or that is to be assessed for the presence of the nucleic acid of interest, and is maintained under conditions which allow hybridization of nucleic acid in the test sample to the oligonucleotides of the array. The array is then exposed to a cleavage agent (single strand or double strand) which cleaves oligonucleotides that are imperfectly hybridized (i.e., not complementary at every location along their sequence) to nucleic acid in the test sample, but does not cleave oligonucleotides that are perfectly hybridized (i.e., that are completely complementary) to nucleic acid in the test sample. The array is then washed to remove any cleavage products, and the pattern of negative and positive signals on the washed, cleaved array is observed. A negative signal corresponds to an imperfectly hybridized oligonucleotide that was cleaved by the cleavage agent (thus removing the label), and a positive signal corresponds to an oligonucleotide that was not cleaved by the cleavage agent.
The assays of the invention allow fully automated high density array or chip-based analysis with a variety of nucleic acid markers, including nucleic acid sequence elements derived from highly polymorphic short sequence repeat markers and markers forming a high density of polymorphisms, such as single nucleotide polymorphisms. The assays allow quick, highly informative assessment of genetic polymorphisms and alterations, thereby facilitating analysis and/or genotyping of a nucleic acid sample. If the genetic polymorphism is an allele-specific polymorphism representing disease-associated alterations, then the assay allows quick, highly informative assessment of disease gene status. The assay also allows quick, highly informative assessment of nucleic acids for the presence of any previous known or unknown alterations in sequence.